The RAG1 protein is part of a multi-functional complex that carries out V(D)J recombination, a programmed DNA rearrangement that is required for development of the immune system in humans. Either alone or in the context of its recombinase partner RAG2, RAG1 encompasses multiple biochemical activities, including DNA binding, DNA cleavage, and ubiquitin ligation. Ubiquitin conjugation is involved in regulation of nearly every biochemical system in eukaryotes, and can mark proteins for degradation by the 26S proteasome or alter their biological properties. Ubiquitin ligases such as RAG1 choose the targets of ubiquitylation, suggesting that RAG1 may exert a regulatory influence over recombination and lymphocyte development. It has been shown previously that mutations in the RAG1 ubiquitin ligase domain are associated with severe immune deficiency in human patients. Consistent with this, we have found that disruption of ubiquitin ligase activity leads to a reduction in recombination in vitro under conditions where RAG1 is limiting, and the isolated RAG1 E3 domain can promote ubiquitylation of several substrates in cell-free systems. The specific hypothesis of this proposal is that RAG1 ubiquitin ligase activity promotes V(D)J recombination during lymphocyte development. This hypothesis will be tested through a detailed examination of the physiological role(s) of RAG1 E3 activity in lymphocyte development in the mouse, including an examination of ubiquitylation of putative RAG1 substrates. We will develop three separate strains of knock-in mice expressing E3-deficient RAG1 alleles, each of which has been extensively characterized in cell-free and in vitro systems, and two of which correspond to alleles identified in immunodeficient human patients. Analysis of these murine models will be absolutely critical in establishing the role of RAG1 ubiquitin ligase activity in lymphocyte development. PUBLIC HEALTH RELEVANCE: The RAG1 protein is part of a multi-functional complex that carries out V(D)J recombination, a programmed DNA rearrangement that is required for development of the immune system in humans. Inactivating mutations in RAG1 lead to severe combined immune deficiency, which is fatal early in life without treatment, while mis-regulation of RAG1 activity can lead to chromosomal translocations associated with lymphoma. This research will elucidate new mechanisms for activation and control of V(D)J recombination, focusing on a biochemical activity integral to RAG1 which is required to support optimal levels of recombination in vitro.